Project Summary Toxoplasmosis is caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii. 30% of the global human population is chronically infected with Toxoplasma, typically without symptoms. However, reawakening of a dormant infection in immunocompromised patients can lead to life-threatening encephalitis and myocarditis observed in 50% and 10% of AIDS patients, respectively. Infection cannot be cured and the disease is managed by prophylactic drug administration, which has severe toxicity, particularly upon prolonged use as applied in AIDS patients. Thus, there is an urgent need for new drugs. Toxoplasmosis pathology originates in repeated rounds of intracellular replication and emergence of parasites from the host cell. Host cell invasion is therefore essential for the progression of toxoplasmosis. The essentiality of this step makes it an excellent target for new anti-Toxoplasma therapeutics, which is the rationale for this proposal. We have already demonstrated that invasion is completely reliant upon the Ca2+- dependent secretion of both micronemes and rhoptries. Interestingly, the last leg of protein trafficking through the secretory pathway to the micronemes and rhoptries does not appear to rely on Rab or SNARE proteins and therefore the Ca2+-dependent membrane fusion machinery involved in this process is likely unorthodox (i.e. a specific drug target). In support of this hypothesis, we identified unusual Ca2+-responsive proteins (TgDOC2 and three ferlins) acting in Ca2+-mediated exocytosis. Preliminary investigation revealed that they operate on distinct secretion events, which represent different functions and thus highlight a novel pathogenic mechanism. This proposal will establish a mechanistic understanding of Ca2+-dependent secretion and will shed much- needed light on the physiology and pathogenesis of Toxoplasma to effectively exploit this target for future drug design. Hereto we will combine genetic, cell biological and biochemical approaches in three specific aims: 1. Test the hypothesis that, in Toxoplasma, the ferlins are distinct Ca2+-sensors in different exocytic pathways, whereas TgDOC2 functions as a general Ca2+-dependent activator for secretion. This will be accomplished by generating specific mutations in these genes and assessing organelle secretion dynamics 2. Determine the function of different microneme secretion content by SILAC and establish how the different mutants displaying distinct deficiencies in Ca2+-exocytosis correlate with distinct steps along the egress-motility-invasion trajectory. 3. Identify the likely unorthodox membrane fusion machinery in which these Ca2+-sensors and activators function using a TAP pull-down and mass spectrometry approach. Upon successfully completing the proposed work we expect to have resolved the molecular basis of Ca2+-dependent secretion and to understand the complexity of differential microneme and rhoptry secretion events in the parasite's pathogenesis. These findings will provide the basis for rational new drug development, which would represent a novel mechanism of action and would be a major advance toward effective control of opportunistic toxoplasmosis in AIDS patients.